The present invention relates to retroviral pseudo-type vectors having modified surface envelope proteins suitable for cell specific transduction of a selected mammalian cell type (cell targeting), methods for the preparation of the cell-specific retroviral pseudo-type vectors and their use for gene transfer into selected cells.
The aim of somatic gene therapy is the effective transfer of genes or gene fragments with functional homology to a defective gene or of genes or gene fragments with therapeutic effects. Previous experiments and clinical studies of somatic gene therapy have been conducted predominantly on the basis of retroviral murine leukemia viruses (MLV). The host cell region of retroviral vectors is determined by the surface envelope protein (SU) encoded by the env gene. The protein products of the env gene form the outer envelope of the retroviral vector. The SU proteins interact with, ie, bind to, a specific protein (receptor) on the surface of the host cell. The env gene products of, for example, amphotropic MLV enable gene transfer into a great number of different mammalian cells. Generally, both ecotropic and amphotropic MLV vectors transduce all murine (ecotropic) and murine and human cells (amphotropic), respectively, since the receptors targeted by these viruses are ubiquitous. Accordingly, cell-specific gene transfer by means of MLV is not possible.
Host cell specificity, however, is advantageous e.g. for the use in gene therapy since in a gene therapy outside of the organism (ex vivo) (Anderson et al., Science 256 (1992) 808-813; Yu et al., H. Gene Therapy 8 (1997) 1065-1072) laborious purifications of cells may be avoided. For therapeutic, diagnostic or vaccination use in vivo it is desirable to specifically target the retroviral vectors to the desired host cells which are affected by genetic malfunctions or are the therapy target, respectively, and subsequently transfer the therapeutic gene.
A restriction of the host cell range of e.g. the amphotropic MLV has been achieved by modification of the surface envelope protein. One modification of the surface envelope protein has been carried out by fusion to a hormone domain. Transduction of the cells carrying the specific hormone receptor occurred (Kasahara et al., Science 266 (1994) 1373-1375). Further, the surface envelope protein was modified by fusion to a single chain antibody fragment (single chain variable fragment, in the following also referred to as xe2x80x9cscFvxe2x80x9d). The fragment represented the antigen binding domain of an antibody and is a fusion protein composed of the variable domains VH and VL of a monoclonal antibody. Both domains are linked via a glycine and serine oligopeptide [-(ser-gly4)3-gly-)] which enables correct folding of the fusion protein (Huston et al., Methods Enzymol. 203 (1991) 46-88, Whitlow et al., Methods: A companion to Methods Enzymol. 2 (1991) 97-105). All modifications of the MLV surface envelope protein using an scFv carried out so far showed that while binding of the vectors to the host target cell occurred, however, they failed to enter the cell (Russel et al., Nucleic Acid Res. 21 (1993) 1081-1085). Furthermore it is known that the surface envelope protein of the MLV generally does not allow extensive modifications (Cosset et al., J. Virol. 69 (1995) 6314-632). Modifications in which a portion of the binding domain of the MLV-SU protein has been replaced led to incorrect processing and, thus, to defects in transport of the SU protein to the cell surface (Weiss et al., In J. A. Levy (ed.) The Retroviridae 2 (1993) 1-108; Morgan et al., J. Virol 67 (1993) 4712-4721; Russel et al., Nucleic Acids Res. 21 (1993) 1081-1085).
To avoid the problem of necessary modification of the surface envelope protein required for the specific targeting of the desired cell type, the preparation of host cell-specific retroviral vectors has been achieved by pseudotyping of for example MLV envelopes. In this case the viral core is derived from MLV and the viral envelope (SU proteins) is derived from other retroviral vectors. The advantages of pseudotyping of MLV capsids are that the risk of replication-competent retroviruses which might arise is minimized. By using different non-homologous expression plasmids encoding the structural genes gag, pol, and env the risk of recombination is reduced. In this manner it has been shown that MLV capsids having ENV proteins of the simian sarcoma associated virus (Takeuschi et al., Virology 186 (1992) 792-794), the feline leukemia virus (Porter et al., Hum. Gene Ther. 7 (1996) 913-919) and of feline endogenous retroviruses (Vile et al., Virology 180 (1991) 420-424), respectively, may be pseudo typed. Reiser et al. (Proc. Natl. Acad. Sci. USA 93 (1996) 15266-15271) were able to demonstrate the possibility of preparation of HIV/MLV and HIV/VSV-G pseudo-types, respectively. Similarly, the possible incorporation of VSV-G protein (Burns et al., Proc. Natl. Acad. Sci. USA 90 (1993) 8033-8037; Ory et al., Proc. Natl. Acad. Sci. USA 93 (1996) 11400-11406) as well as the incorporation of truncated HIV env glycoproteins (Schnierle et al., Proc. Natl. Acad. Sci. USA 94 (1997) 8640-8645) has been described. The vectors pseudo typed in this way, however, are unable to target any desired cell-specific receptor of a cell type since the target cell is determined only by the tropism (cell-specificity of a virus) of the respective viral surface protein.
Generally, gene transfer into mammalian cells by means of (pseudo typed retroviruses) retroviral vectors has following benefits:
Normally, one copy of the desired gene is transferred into the mammalian cell.
Generally, the desired gene is transferred without mutation or rearrangements.
A stable incorporation of the desired gene into the genome of the target cell occurs.
Furthermore, gene transfer into mammalian cells using pseudo-typed retroviruses has the advantage that by means of pseudo typing e.g. MLV, HIV, foamy virus, or SIV capsids mainly with SNV-ENV protein a specific alteration in cell specificity of the respective retroviral vectors may be achieved so that for example a therapeutical gene may be introduced into a selected cell population.
Therefore, it is an object of the present invention to provide retroviral pseudo type vectors having modified surface envelope proteins suitable for cell-specific transduction of a selected mammalian cell type (cell targeting). Further, it is an object of the present invention to provide a method for the preparation of said cell-specific retroviral pseudo type vectors. Moreover, it is an object of the present invention to provide retroviral packaging cells for obtaining the vectors according to the present invention. In contrast to vectors known up to now, using these vectors it is possible to target any desired receptor of a target cell.
The object of the present invention has been solved by the retroviral vectors of the present invention comprising viral cores of e.g. murine leukemia virus (MLV), human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), or foamy virus as well as viral capsids of spleen necrosis virus (SNV). Preferred vectors are retroviral vectors in which the viral envelopes comprise the full length surface protein (SU protein) of SNV and/or a chimeric SNV-non-viral polypeptide-ENV, SNV-HIV-ENV or SNV-SIV-ENV. Particularly preferred are retroviral vectors in which the non-viral polypeptide comprises a ligand, a peptide fragment of a ligand, an antibody, a peptide fragment of an antibody or an antibody recognition domain (scFv). Further preferred are retroviral vectors which further comprise an RNA to be introduced into the cell which is to be transduced by the retroviral vector. Particularly preferred are retroviral vectors in which the RNA comprises a therapeutic gene or a nucleic acid fragment of a therapeutic gene and/or a reporter gene. Especially preferred are retroviral vectors wherein the therapeutic gene or the nucleic acid fragment of a therapeutic gene comprises the CFTR gene, phox91, ADA, IL-16, p53, transdominant mutants (e.g. revM10) as well as vaccination genes, e.g. recombinant gp120 and IL-16. Further particularly preferred are retroviral vectors wherein the reporter gene comprises xcex2-galactosidase, xe2x80x9cgreen fluorescent proteinxe2x80x9d, luciferase or the resistance genes neomycin or xe2x80x9cmultiple drug resistance genexe2x80x9d. The retroviral vectors according to the present invention may be used as medicaments. The use in the preparation of a medicament for somatic gene therapy, vaccination therapy or diagnostics is preferred. Particularly preferred is the therapy of cystic fibrosis, ADA deficiency, chronic granulomatosis, and HIV infection.
Furthermore, the object of the present invention has been solved by the retroviral packaging cells according to the invention for obtaining the retroviral vectors of the invention. The retroviral packaging cells according to the present invention are transformed both with one or more psi-negative expression construct(s) expressing the gag and pol gene products of MLV, HIV, SWV, or foamy virus, and with a psi-negative SNV-Env and/or psi-negative SNV-Env-non-viral polypeptide, psi-negative SNV-HIV-ENV or SNV-SIV-env expression construct. A retroviral packaging cell is preferred in which the non-viral polypeptide of the psi-negative SNV-Env-non-viral polypeptide expression construct comprises a ligand, a peptide fragment: of a ligand, an antibody, a peptide fragment of an antibody or an antibody recognition domain (scFv). Further preferred is a retroviral packaging cell line further comprising a psi-positive expression construct comprising a nucleic acid sequence to be introduced into the cell to be transduced by the retroviral vector. Especially preferred is a retroviral packaging cell line, wherein the nucleic acid sequence comprises a therapeutic gene or its nucleic acid fragment and/or a reporter gene. Particularly preferred is a retroviral packaging cell line wherein the therapeutic gene or the nucleic acid fragment of a therapeutic gene comprises the CFTR gene, phox91, ADA, IL-16, p53, transdominant mutations (e.g. revM10), and vaccination genes, e.g. recombinant gp120 and IL-16. Further particularly preferred is a retroviral packaging cell line wherein the reporter gene comprises xcex2-galactosidase, xe2x80x9cgreen fluorescent proteinxe2x80x9d, luciferase or the resistance genes neomycin or the xe2x80x9cmultiple drug resistance genexe2x80x9d.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.